The hypothesis that transformation of human bronchial epithelial cells (NHBE) following transfection with a v-Ha-ras containing plasmid includes an activity that interferes with the maintenance of normal chromosome was tested. The "karyotypic instability" hypothesis was examined by spreading chromosomes of mitotic cells after protoplast fusion transfection of NHBE cells from several donors with a plasmid (h1) that carries v-Ha-ras. The effects of transient expression of transfectes sequences on the population were tested immediately after transfection and one passage later. The results were scored as total chromosomal abnormalities and the observations from four experiments summarized as follows: 1) v-Ha-ras transfected NHBE cells, 45/268 mitosis (16.8%); 2) pBR322 transfected NHBE cells, 8/200 mitosis (4.0%); 3) protoplast fusion no plasmid treated NHBE cells (7.0%); 4) untreated NHBE cells, 17/200 mitosis (8.5%). Thus, a significant increase in chromosomal abnormalities occurs in NHBE cell populations transfected by v-Ha-ras. This effect was observed 24 to 72 hours after introduction of the Ha-raw oncogene, during the period preceding the selection of transformed populations of NHBE cells by challenge with growth conditions that stimulate terminal differentiation of NHBE cells (i.e., 2-4% serum added to serum free LHC-medium). This increase in karyotypic instability is potentially important to the mechanism of v-Ha-ras transformation of NHBE cells and is consistent with the observed requirement for secondary rare events for survival of senescence, establishment of indefinite culture life span, and tumorigenicity of transformed NHBE cells.